The present invention relates to compounds and their use for the treatment of nuclear hormone receptor (NHR) family associated disorders. More specifically, the present invention relates to compounds having a particular 3-dimensional spatial orientation that are capable of binding to and thus altering the function of NHRs. Such compounds would be useful as therapeutic agents for disorders associated with NHRs such as the retinoid x receptor (RXR). The invention also relates to compositions and methods for the treatment or prophylaxis of osteoporosis, bone loss, arthritis, inflammation, cancer and skin conditions.
Modern day methods for the discovery of therapeutic agents for the amelioration of major diseases center on the interdisciplinary approaches of molecular biology, enzymology, crystallography, drug synthesis, molecular modeling and pharmacology. The typical approach involves: identification, isolation, purification, and crystallization of a target protein associated with the disease(s) of interest; modeling the protein binding and active sites; and modeling, synthesizing and evaluating compounds to optimize their pharmacological activity. Even with the advanced state of the art, drug discovery and, in particular, prediction of structure-activity relationships continue to require significant effort on the part of the pharmaceutical industry. Thus, the need to develop efficient and cost effective methods for the identification of pharmacologically active compounds for the treatment specific diseases still remains.
Osteoporosis is a condition characterized by a decrease in bone mass with decreased density and enlargement of bone spaces, producing porosity and fragility. This condition afflicts both men and women, particularly menopausal women, with advancing age. This condition is primarily a disorder in the formation of bone matrix. Osteoblasts, the bone-forming population of cells, are typically reduced in number. Osteoblasts are derived from adjacent mesenchymal precursors in a process regulated by local bone-derived factors. Osteoclasts, a population of cells that break down bone and that are associated with bone resorption, are not reduced in number. Osteoclasts are large, usually multinuclear cells found on the resorbing surfaces of mineralized bone. Osteoclasts are formed by fusion of mononuclear precursors, originating from extraskeletal blood-born precursors.
All known and local stimulators of osteoclastic bone resorption, including parathyroid hormone, 1, 25D, IL-2, and TNF, modulate their stimulatory effects on the osteoclast through an initial effect on osteoblasts. Osteoblasts are therefor believed to play a major role in regulating bone turnover by controlling the rate of new bone formation, as well as by serving to generate signals that stimulate osteoclastic bone resorption.
NHR families are associated with the modulation of mammalian cell proliferation and differentiation. These cellular processes are controlled by signal molecules that regulate gene expression. NHRs such as retinoid receptors are associated with many diseases and disorders such as osteoporosis, cancer, acne, AIDS, arthritis, psoriasis, lupus erythematosus and the like. The retinoid x receptor (RXR) serves to modulate cellular transcriptional activity thereby controlling cellular proliferation.
It is recognized in the art that osteoblasts play a very complex role in the formation of bone. It is generally thought that osteoclasts serve to dissolve (resorb) bone so that osteoblasts can then deposit more bone. It is reasonable then that compounds which can either inhibit the excessive resorption of bone or stimulate the proliferation of osteoblasts will be useful for the prevention of bone loss or the stimulation of bone growth.
Takashi et al. (Jpn. Kokai Tokyo Koho JP03130216 A2, Jun. 4, 1994) discloses diphenyl compounds having the following general structure 
where X is xe2x80x94CH2xe2x80x94 or xe2x80x94C(xe2x95x90O)xe2x80x94, for the treatment and prophylaxis of osteoporosis.
Labroo (U.S. Pat. No. 5,389,646 issued Feb. 14, 1995) discloses compounds having the following general structure 
where R1 is H, OH, C1-C17 alkoxy, (C1-C17)alkylcarbonyloxy, (C1-C17)alkylcarbonylamino or (C1-C17)alkylcarbonyl; R2 is xe2x80x94(CH2)(1-6)xe2x80x94CH2-heterocycle; and R3 is H, OH, C1-C17 alkoxy, (C1-C17)alkylcarbonyloxy, (C1-C17)alkylcarbonylamino or (C1-C17)alkylcarbonyl, for the treatment and prevention of bone loss.
Other compounds such as the one below 
have been proposed for the treatment of osteoporosis. Even so, those compounds have not found general use due to their limited efficacy. Thus, the need for more efficacious compounds for the treatment of osteoporosis still remains.
Kamala et al. (Indian J. Chem. (1983), 22B, 1194-96) and Waisser et al. (Collect. Czech. Chem. Commun. (1991), 56, 2978-2985) disclose the synthesis of 
and its uses as a synthetic intermediate and anti-tuberculotic agent, respectively.
Bis-aromatic compounds are widely known for their use in the treatment of cancer and tumors. Such compounds generally effectively inhibit DNA replication thereby exerting their cytotoxic effect upon mammalian cells. Here too, no compound has been found to be generally applicable for the treatment of a broad spectrum of cancers and tumors. Thus, the need for more efficacious broader spectrum anticancer and tumoricidal compounds still remains.
It is an object of the present invention to overcome the limitations inherent in the art of modeling pharmaceutical agents by providing a method for selecting candidate chemical agents using defined 3-dimensional spatial characteristics. These spatially defined chemical compounds in some aspects further overcome limitations associated with available osteogenic agents by providing agents that stimulate production of bone morphogenic proteins, and thus provide compositions useful for stimulating osteoblast proliferation and in diseases which result in bone loss.
A further object of the invention is to provide agents that bind other receptors in the nuclear hormone receptor (NHR) family that are associated with diseases.
It is another object of the present invention to provide a method of using particular pharmacologically active compounds for the treatment or prophylaxis of physiological disorders or diseases associated with NHRs such as osteoporosis, arthritis, cancer, tumors and the like.
It is another object of the invention to provide a method for the selection of pharmacologically active compounds which are capable of stimulating osteoblast proliferation and differentiation and are useful for the treatment of physiological disorders associated with NHRs and in particular diseases associated with bone loss.
Another object of the present invention is to provide methods for selecting and screening for pharmacologically active compositions which are capable of stimulating osteoblast proliferation and differentiation activity. Such selected composition would be used for the treatment or prophylaxis of osteoporosis and other physiological disorders associated with NHRS.
The present invention provides methods of stimulating osteoblast proliferation, as well as methods for selecting pharmacologically active compounds. In one embodiment, the method for stimulating osteoblast proliferation comprises selecting substances of the general formula I
Xxe2x80x94Lxe2x80x94Z,
wherein:
X is selected from the group consisting of: 
L is selected from the group consisting of: 
Z is selected from the group consisting of: 
wherein R1 is selected from the group consisting of:
H, OH, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylthio, halo and (C1-C12)alkyl-carbonyloxy;
R2 is selected from the group consisting of:
H, OH, halo, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkoxy and (C1-C12)alkyl-carbonyloxy;
R3 is selected from the group consisting of:
H, OH, halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkenyl and (C1-C12)alkyl-carbonyloxy;
R4 is selected from the group consisting of:
H, OH, halo, C1-C6 alkyl, C1-C6 alkoxy and (C1-C12)alkyl-carbonyloxy;
R5 is selected from the group consisting of:
H, halo, C1-C6 alkyl, C1-C6 alkoxy, xe2x80x94OC(xe2x95x90O)Me, phthalimide and (C1-C12)alkyl-carbonyloxy;
R6 is selected from the group consisting of:
H, OH, xe2x80x94NH2, C1-C4 alkyl and C1-C4 alkoxy;
R7 is selected from the group consisting of:
H, C1-C4 alkyl, (C1-C4)alkyl-carbonyl and (C7-C10)arylalkyl;
R8 is selected from the group consisting of:
H, OH, halo, xe2x80x94CF3, C1-C4 haloalkyl, C1-C4 alkyl, C1-C4 alkoxy, xe2x80x94NHC(xe2x95x90O)Me and xe2x80x94N(C1-C4 alkyl)2;
R9 is selected from the group consisting of:
H, OH, halo, xe2x80x94CN, xe2x80x94NO2, C1-C4 haloalkyl, xe2x80x94CF3, C1-C8 alkyl, C1-C8 alkoxy, xe2x80x94NHC(xe2x95x90O)Me and xe2x80x94OC(xe2x95x90O)Me;
R10 is selected from the group consisting of:
H, OH, halo, xe2x80x94CN, xe2x80x94NO2, C1-C4 haloalkyl, xe2x80x94CO2H, C1-C12 alkyl, C1-C12 alkoxy, phenyl, C1-C12 alkenyl, (C1-C4)alkoxycarbonyl, xe2x80x94NHC(xe2x95x90O)Me, (C1-C4)alkylcarbonyl, (C1-C12)alkylcarbonyloxy and heteroaryl;
R11 is selected from the group consisting of:
H, OH, halo, C1-C4 haloalkyl, xe2x80x94CF3, C1-C4 alkyl, xe2x80x94NH2, C1-C4 alkoxy, xe2x80x94NHC (xe2x95x90O)Me, C1-C4 alkenyl, (C1-C4)alkoxycarbonyl, (C1-C4)alkylcarbonyl, and (C1-C4)alkylcarbonyloxy;
R12 is selected from the group consisting of:
H, OH, xe2x80x94NR2, C1-C4 alkyl, C1-C4 alkoxy, and (C1-C4 alkylcarbonyl; and
R13 is selected from the group consisting of:
H, OH, halo, xe2x80x94NH2, C1-C4 alkyl, C1-C4 alkoxy, xe2x80x94N(C1-C4 alkyl)2; and
exposing cells comprising osteoblast cells to an effective amount of the pharmacologically active compound.
These methods are expected to result in the stimulation of osteoblast proliferation, and hence a useful application in the prevention of bone loss and/or promotion of bone growth.
In further defined embodiments, the substance selected as part of the method is more particularly defined in that
R10 and R11 may join together to form a 5-7 membered carbocycle or oxacarbocycle fused to the ring to which they are attached, where the carbocycle and oxacarbocycle are substituted with one or more groups independently selected from the group consisting of:
C1-C4 alkyl, C1-C4 alkoxy, OH, halo, carboxyl, H and aryl, to provide a pharmacologically active compound.
It is also contemplated and within the scope of the invention that the pharmacologically active compound of the formula I may bind more than one different type of nuclear hormone receptor (NHR), and hence be useful in treatment of other NHR related diseases.
While not intending to be limited to any particular mechanism of action, it is contemplated that the pharmacologically active compound of the formula I may promote osteoblast growth or enrich osteoblast population by enhancing BMP-2 promoter activity.
It is also contemplated and within the scope of the present invention that pharmacologically active compounds of the formula I may be used in combination with other compounds for the stimulation or promotion of osteoblast growth. They may also be used for, the stimulation of bone growth or the inhibition of bone loss and/or bone resorption.
Another aspect of the present invention provides a method for selecting a pharmacologically active compound or candidate pharmacologically active compounds. In one embodiment, the pharmacological activity of the compounds is a capacity for stimulating BMP-2 promoter activity. The method in one embodiment comprises:
selecting candidate compounds having a spatially defined 3-dimensional structure as in formula II
Wxe2x80x94Lxe2x80x94Yxe2x80x83xe2x80x83Formula II
wherein:
W contains an aromatic group having a centroid indicated by the letter xe2x80x9cAxe2x80x9d;
Y contains a carbocyclic group having a centroid indicated by the letter xe2x80x9cBxe2x80x9d;
L is a group linking X and Z;
a plane xe2x80x9cPxe2x80x9d is formed by the aromatic atoms of the aromatic group in W;
the centroid xe2x80x9cBxe2x80x9d lies within about 1, or about 0.7 angstroms above or below the plane xe2x80x9cPxe2x80x9d;
the centroid xe2x80x9cAxe2x80x9d and the centroid xe2x80x9cBxe2x80x9d are spaced apart by about 6, or about 6.6, to about 8, or about 8.5 angstroms;
to provide spatially defined molecules; and
selecting spatially defined molecules capable of stimulating BMP-2 promoter activity.
The spatially defined molecules in the above method are further defined in some embodiments as having at least two hydrogen bond accepting groups located either within or in close proximity to L, the hydrogen bond accepting groups being further defined as follows:
1) the hydrogen bond accepting groups are within about 2, or about 2.3, to about 5, or about 5.4 angstroms apart;
2) one hydrogen bond accepting group is about 4, or about 4.5, to about 7, or about 7.7 angstroms from centroid xe2x80x9cAxe2x80x9d and about 2, or about 2.7, to about 3, or about 3.8 angstroms from centroid xe2x80x9cBxe2x80x9d; and
3) one hydrogen bond accepting group is about 2, or about 2.6, to about 3, or about 3.8 angstroms, from centroid xe2x80x9cAxe2x80x9d and about 4, or about 4.6, to about 7, or about 6.9 angstroms from centroid xe2x80x9cBxe2x80x9d.
In some embodiments of the method, L is further defined as occupying a space which outer limit is less than or equal to about 3, preferably about 3.1 angstroms, as measured by heavy atom distance, above or below and normal to the plane xe2x80x9cPxe2x80x9d as measured along a normal to the plane xe2x80x9cPxe2x80x9d. In other embodiments, L may be even further defined as occupying a space which outer limit is about 4, preferably about 4.7 to about 6.0 angstroms, as measured by heavy atom distance, perpendicular to a line connecting centroid xe2x80x9cAxe2x80x9d to centroid xe2x80x9cBxe2x80x9d and within the plane xe2x80x9cPxe2x80x9d.
Pharmaceutically acceptable preparations of these compounds are also claimed, and are used to provide a pharmacologically active preparation for stimulating osteoblast proliferation and/or reducing bone resorption.
It is contemplated and within the scope of the present invention that the pharmacologically active compound of the formula II may possess activities other than stimulation of osteoblast proliferation and inhibiting bone resorption. Such activities would generally be associated with binding to one or more nuclear hormone receptors, and in so doing providing a treatment for various other diseases that are associated with receptor activity.
It is also contemplated and within the scope of the present invention that many pharmacologically active compounds not specifically enumerated herein may nonetheless fall within the spatially defined structure of formula II. Such spatially defined molecules would also fall within the scope of the method, where they are capable of stimulating BMP-2 promoter activity, or stimulating osteoblast proliferation, or some other desired pharmacological activity. These compounds thus would have utility in stimulating bone formation or reducing the loss of bone tissue.
It should be understood that a compound of the formula II may also contain other structural parameters not recited herein, yet fall within the defined scope of the invention. Such other structural parameters are generally secondary to those recited. That is, although addition of a particular substituent to either W or Y may render a compound of formula II more or less active than without that particular substituent, the compound having the substituent is within the scope of the present method as long as it comprises the characteristics recited above for the compound of formula II, or any of the formulas as described here.
Another embodiment of the present invention provides a method for treating and/or preventing malignant cell proliferation one embodiment of the method comprises:
administering to a population of cells comprising malignant cells a malignant cell proliferation inhibiting amount of a pharmacologically active compound having a spatially defined structure as defined by formula II
Wxe2x80x94Lxe2x80x94Y; andxe2x80x83xe2x80x83Formula II
xe2x80x83inhibiting malignant cells, wherein the pharmacologically active compound is further defined by a 3-dimensional structure wherein:
W contains an aromatic group having a centroid indicated by the letter xe2x80x9cAxe2x80x9d;
Y contains a carbocyclic group having a cenzvroid indicated by the letter xe2x80x9cBxe2x80x9d;
L is a group linking X and Z;
a plane xe2x80x9cPxe2x80x9d is formed by the aromatic atoms of the aromatic group in W;
the centroid xe2x80x9cBxe2x80x9d lies within about 1, or about 0.7 angstroms above or below the plane xe2x80x9cPxe2x80x9d, and
the controid xe2x80x9cAxe2x80x9d and the centroid xe2x80x9cBxe2x80x9d are about 6, or 6.6 to about 8, or about 8.5 angstroms apart.
The pharmacologically active compound of the above method may be further defined as having a spatially defined structure wherein at least two hydrogen bond accepting groups are located either within or in close proximity to L, with the hydrogen bond accepting groups being further defined as follows;
1) the hydrogen bond accepting groups are within about 2, or about 2.3, to about 5, or about 5.4 angstroms apart;
2) one hydrogen bond accepting group is about 4, or about 4.5, to about 7, or about 7.7 angstroms from centroid xe2x80x9cAxe2x80x9d and about 2, or about 2.7, to about 3, or about 3.8, angstroms from centroid xe2x80x9cBxe2x80x9d; and
3) one hydrogen bond accepting group is about 2, or about 2.6, to about 3, or about 3.8, angstroms from centroid xe2x80x9cAxe2x80x9d and about 4, or about 4.6 to about 6, or about 6.9 angstroms from centroid xe2x80x9cBxe2x80x9d.
In some embodiments of the method, L is further defined as occupying a space which outer limit is less than or equal to about 2.5, or about 3 or 3.1 angstroms, as measured by heavy atom distance, above or below and normal to the plane xe2x80x9cPxe2x80x9d as measure along a normal to the plane xe2x80x9cPxe2x80x9d. In yet another embodiment, the pharmacologically active compound is further defined wherein L occupies a space which outer limit is about 4, or preferably about 4.7 to about 5, or preferably 6.0 angstroms, as measured by heavy atom distance, perpendicular to a line connecting centroid xe2x80x9cAxe2x80x9d to centroid xe2x80x9cBxe2x80x9d and within the plane xe2x80x9cPxe2x80x9d.
Other aspects of the present invention provide methods of stimulating BMP-2 promoter activity or osteoblast proliferation and pharmacologically active compounds of the formula III 
wherein:
R1 is selected from the group consisting of:
aryl, naphthyl, heteroaryl, cycloalkyl, cycloalkenyl, azacycloalkyl, oxacycloalkyl, azacycloalkenyl, oxacycloalkenyl, keto substituted cycloalkyl, and keto substituted cycloalkenyl, where each of the above substituents is substituted by one or more of the groups independently selected from the group consisting of:
C1-C7 alkyl, C1-C7 alkoxy, benzyloxy, hydroxy, C1-C2 haloalkyl, halo, cyano, carboxyl, hydrogen, (C1-C4)alkoxycarbonyl, xe2x80x94N(C1-C4 alkyl)2, (C1-C4)alkylcarbonyloxy, aryl, (C1-C4)alkylcarbonylamino, (C1-C4)alkylcarbonyl, (C1-C4)alkyl-aryl, and xe2x80x94NH2;
R2 is selected from the group consisting of:
H, C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkynyl, C1-C4 alkoxy and xe2x80x94NH2;
R3 and R6 are selected from the group consisting of:
H, hydroxy, halo, (C1-C5)alkylcarbonyloxy, cyano, C1-C4 alkyl, C1-C4 alkenyl and C1-C4 alkoxy;
R4 and R5 are selected from the group consisting of:
H, halo, hydroxy, (C1-C4)alkyl-carbonyloxy, cyano, C1-C2 haloalkyl, C1-C4 alkoxy, benzoyl, (C1-C4)alkyl-aryl, (C1-C6)alkylaminocarbonyloxy, phenylaminocarbonyloxy, C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkynyl, (C1-C4)alkenyl-aryl, (C1-C4)alkynyl-aryl, (C1-C4)alkyl-(C6-C10)cycloalkyl, (C1-C4)alkenyl-(C6-C10)cycloalkyl, (C1-C4)alkynyl-(C6-C10)cycloalkyl, (C1-C4)alkyl-(C6-C10)cycloalkenyl, (C1-C4)alkenyl-(C6-C10)cycloalkenyl, (C1-C4)alkynyl-(C6-C10)cycloalkenyl, carboxy and (C1-C4)alkoxycarbonyl.
The method generally comprises administering an effective amount of a compound of the formula III to cells comprising osteoblasts or osteoblast precursor cells.
Further defined embodiments of the method for stimulating BMP-2 promoter activity or osteoblast proliferation employ compounds of the type described above wherein R3 and R4 join together to form a 5-7 membered carbocycle or oxacarbocycle fused to the ring to which they are attached, where the carbocycle or oxacarbocycle is substituted by one or more of the groups selected from the group consisting of: C1-C4 alkyl, C1-C4 alkoxy, hydroxy, halo, carboxyl, hydrogen and aryl.
In alternative embodiments of the method, the pharmacologically active compound is defined as follows:
R4 and R5 join together to form a 5-7 membered carbocycle or oxacarbocycle fused to the ring to which they are attached, where the carbocycle or oxacarbocycle is substituted by one or more of the groups selected from the group consisting of: C1-C4 alkyl, C1-C4 alkoxy, hydroxy, halo, carboxyl, hydrogen and aryl.
In yet another alternative embodiment, the pharmacologically active compound is further defined wherein:
R5 and R6 join together to form a 5-7 membered carbocycle or oxacarbocycle fused to the ring to which they are attached, where the carbocycle or oxacarbocycle is substituted by one or more of the groups selected from the group consisting of: C1-C4 alkyl, C1-C4 alkoxy, hydroxy, halo, carboxyl, hydrogen and aryl.
Another aspect of the present invention comprises a pharmacologically active composition possessing osteoblast proliferation activity. In some embodiments, the composition is defined by reference to a particularly defined process comprising:
oxidizing a compound of the formula VI 
with either heat or CrO3/H2O/AcOH for a period of time sufficient to form the pharmacologically active composition. This preparation may be further purified and the most active fractions selected. Compositions provided as a result of the above process and having an ED50 of about 1 to about 50 xcexcg/ml, as measured using the assay as described in Example 1, are expected to possess osteoblast proliferation activity and a capacity for stimulating BMP-2 promoter activity.
It is contemplated and within the scope of the present invention that the above process may comprise other similar or equivalent processes which will effect the conversion of the compound of the formula VI to a desired composition having the defined pharmacological activity. Such methods do not depart from the spirit or scope of the present invention.
It is also contemplated and within the scope of the present invention that the pharmacologically active composition so prepared will be useful in the treatment or prophylaxis of diseases or disorders associated with tumor cell proliferation, arthritis, inflammation, bone resorption, skin conditions, and diseases that may be treated employing receptor-directed therapies.
Other features, advantages and embodiments of the invention will be apparent to those skilled in the art from the following description, accompanying data and appended claims.